Riboswitch-inspired toehold riboregulators for gene regulation in Escherichia coli

Regulatory RNA molecules have been widely investigated as components for synthetic gene circuits, complementing the use of protein-based transcription factors. Among the potential advantages of RNA-based gene regulators are their comparatively simple design, sequence-programmability, orthogonality, and their relatively low metabolic burden. In this work, we developed a set of riboswitch-inspired riboregulators in Escherichia coli that combine the concept of toehold-mediated strand displacement (TMSD) with the switching principles of naturally occurring transcriptional and translational riboswitches. Specifically, for translational activation and repression, we sequestered anti-anti-RBS or anti-RBS sequences, respectively, inside the loop of a stable hairpin domain, which is equipped with a single-stranded toehold region at its 5' end and is followed by regulated sequences on its 3' side. A trigger RNA binding to the toehold region can invade the hairpin, inducing a structural rearrangement that results in translational activation or deactivation. We also demonstrate that TMSD can be applied in the context of transcriptional regulation by switching RNA secondary structure involved in Rho-dependent termination. Our designs expand the repertoire of available synthetic riboregulators by a set of RNA switches with no sequence limitation, which should prove useful for the development of robust genetic sensors and circuits.